Although the cystic fibrosis genotype is associated with defective plasma membrane Cl conductance, little is known about the cellular abnormality that links genotype to clinical disease. Recent observations focus attention on trafficking defects in cystic fibrosis: trafficking of CFTR and of endosomal and secretory vesicles, and defective acidification. We propose to apply novel fluorescence labeling and imaging techniques to test definitively whether vesicular trafficking and/or acidification constitute major cellular defects in cystic fibrosis, and to determine whether CFTR is multi-functional water-filled channel. Specific Aim 1. To define the mechanisms of acidification in vesicular compartments of the endosomal and secretory pathways in epithelial cells which contain and do not contain CFTR. - Fluorescence labeling and imaging techniques will be developed to obtain the first information about acidification regulatory mechanisms in the endosomal and secretory compartments of epithelial cells. Novel technical developments include the synthesis of pH and Cl-sensitive indicators for labeling trans-Golgi and secretory vesicles in living cells, and application of quantitative confocal imaging methods to measure pH and Cl activity in fluorescently- labeled vesicles in living cells in real time. Specific Aim 2. To test the hypothesis that cystic fibrosis is associated with defective acidification and/or trafficking in the endosomal and secretory compartments. - New fluorescence methods will be applied to compare vesicular acidification mechanisms and the kinetics of endocytosis and exocytosis in normal and cystic fibrosis cells. The subcellular distribution of functional Cl channels will be determined by Cl transport measurements in isolated subcellular vesicles. Primary and transfected cell cultures will be used to compare the normal vs. cystic fibrosis phenotype. Specific Aim 3. To determine whether CFTR contains a cAMP-gated aqueous pathway for transport of not only Cl, but of water and selected solutes. - Preliminary studies show that CFTR expression in Xenopus oocytes is associated with a cAMP-stimulated anion conductance and water conductance. Xenopus oocyte and mammalian cell expression systems will be used to define the route and mechanism(s) of the increased water permeability. The data should provide: a. definitive evidence to support or refute the acidification and trafficking hypotheses, b. information on novel intracellular role(s) of CFTR and c. information on mechanisms of acidification and trafficking in epithelial cell endosomal and secretory compartments.